RESUMO
A series of inhibitors of Pim-2 kinase identified by high-throughput screening is described. Details of the hit validation and lead generation process and structure-activity relationship (SAR) studies are presented. Disclosure of an unconventional binding mode for 1, as revealed by X-ray crystallography using the highly homologous Pim-1 protein, is also presented, and observed binding features are shown to correlate with the Pim-2 SAR. While highly selective within the kinase family, the series shows similar potency for both Pim-1 and Pim-2, which was expected on the basis of homology, but unusual in light of reports in the literature documenting a bias for Pim-1. A rationale for these observations based on Pim-1 and Pim-2 K(M(ATP)) values is suggested. Some interesting cross reactivity with casein kinase-2 was also identified, and structural features which may contribute to the association are discussed.
Assuntos
Azepinas/química , Modelos Moleculares , Fenilpropionatos/química , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/química , Azepinas/síntese química , Sítios de Ligação , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/química , Cristalografia por Raios X , Fenilpropionatos/síntese química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Benzimidazole 1 was identified as a selective inhibitor of ITK by high throughput screening. Hit-to-lead studies defined the SAR at all three substituents. Reversing the amide linkage at C6 led to 16, with a fivefold improvement of potency. This enhancement is rationalized by the conformational preference of the substituent. A model for the binding of the benzimidazoles to the ATP-binding site of ITK is proposed.
Assuntos
Benzimidazóis/química , Química Farmacêutica/métodos , Inibidores de Proteínas Quinases/síntese química , Proteínas Tirosina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/química , Benzimidazóis/síntese química , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Humanos , Ligação de Hidrogênio , Concentração Inibidora 50 , Modelos Químicos , Ligação Proteica , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
Numerous assay methods have been developed to identify small-molecule effectors of protein kinases, but no single method can be applied to all isolated kinases. The authors developed a set of 3 high-throughput screening (HTS)-compatible biochemical assays that can measure 3 mechanistically distinct properties of a kinase active site, with the goal that at least 1 of the 3 would be applicable to any kinase selected as a target for drug discovery efforts. Two assays measure catalytically active enzyme: A dissociation-enhanced lanthanide fluoroimmuno assay (DELFIA) uses an antibody to quantitate the generation of phosphorylated substrate; a second assay uses luciferase to measure the consumption of adenosine triphosphate (ATP) during either phosphoryl-transfer to a peptide substrate or to water (intrinsic ATPase activity). A third assay, which is not dependent on a catalytically active enzyme, measures the competition for binding to kinase between an inhibitor and a fluorescent ATP binding site probe. To evaluate the suitability of these assays for drug discovery, the authors compared their ability to identify inhibitors of a nonreceptor protein tyrosine kinase from the Tec family, interleukin-2-inducible T cell kinase (ITK). The 3 assays agreed on 57% of the combined confirmed hit set identified from screening a 10,208-compound library enriched with known kinase inhibitors and molecules that were structurally similar. Among the 3 assays, the one measuring intrinsic ATPase activity produced the largest number of unique hits, the fewest unique misses, and the most comprehensive hit set, missing only 2.7% of the confirmed inhibitors identified by the other 2 assays combined. Based on these data, all 3 assay formats are viable for screening and together provide greater options for assay design depending on the targeted kinase.
Assuntos
Adenosina Trifosfatases/metabolismo , Bioensaio/métodos , Inibidores de Proteínas Quinases/análise , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Corantes Fluorescentes/química , Humanos , CinéticaRESUMO
The tyrosine kinase p56lck (lck) is essential for T cell activation; thus, inhibitors of lck have potential utility as autoimmune agents. Our initial disclosure of a new class of lck inhibitors based on the phenylaminoimidazoisoquinolin-9-one showed reasonable cellular activity but did not work in vivo upon oral administration. Our current work highlights the further use of rational drug design and molecular modeling to produce a series of lck inhibitors that demonstrate cellular activity below 100 nM and are as efficacious as cyclosporin A in an in vivo mouse model of anti-CD3-induced IL-2 production.